人心肌干细胞的体外改良培养

Culture of human cardiac stem cells with improved cardiosphere-growing medium in vitro

目的:用改良的培养基培养人心肌干细胞,并筛选鉴定。方法:通过心脏外科手术取下右心耳组织,用组织块培养法来获得原代心肌干细胞,用改良的心肌干细胞培养液培养,增殖传代后进行心肌干细胞表面标志物的鉴定,再用免疫磁珠筛选以获得较纯的目的心肌干细胞。结果:培养约2周后,贴壁良好的心肌组织块周围可见有小、圆、亮的细胞爬出,用Accutase(细胞消化液)短时间消化后冲洗细胞,培养增殖传代,其增殖能力与传统心肌干细胞培养液培养的细胞比较无显著差异,用流式细胞术鉴定c-Kit(干细胞表面标志物),其阳性率(6.8±2.1)%,之后用含抗c-Kit抗体(anti-c-Kit)的磁珠进行分选,即得较纯的c-Kit阳性(c-Kit+)心肌干细胞,它们可以分化为心肌细胞。结论:通过心脏组织块贴壁培养法,用改良后的心肌干细胞培养基培养,再借助免疫磁珠的分选,同样可得到较纯的c-Kit+心肌干细胞。

AIM： To prepare an improved medium for culturing human cardiac stem cells. METHODS： The heart samples of the right auricle obtained from the patients after cardiac surgery were minced into pieces （ about 1 mm x 1 mm x 1 mm）, digested and cultured. The primary cells obtained were cultured with improved cardiosphere-growing medium （CGM） for proliferation, and the cells were identified by flow cytometry. Finally, purer c-kit ~ cells were obtained by the method of magnetic bead sorting. RESULTS ： After about 2 weeks of culture, small, round and phase-bright cells migrated from the well-adherent explants over a layer of fibroblast-like ceils. These cells were collected by a brief digestion with Ac- cutase, washed and cultured with improved CGM. No significant difference of the proliferative capacity between using tradi- tional CGM and improved CGM was observed. After subculture and proliferation, the identification result by flow cytometry showed that the positive rate of c-Kit surface marker on these cells was （6.8 ＋ 2.1 ） %. By the method of anti-c-Kit mag- netic bead sorting, purer c-Kit ~ cardiac stem cells were obtained and differentiated into cardiomyocytes. CONCLUSION： Purer c-Kit ~ cardiac stem cells are isolated with the improved CGM culture.